ReViTA: A novel in vitro transcription system to study gene regulation

ReViTA (Reverse in VitroTranscription Assay) is a novel in vitro transcription-based method to study gene expression under the regulation of specific transcription factors. The ReViTA system uses a plasmid with a control sequence, the promoter region of the studied gene, the transcription factor of interest, and an RNA polymerase saturated with σ70. The main objective of this study was to evaluate the method; thus, as a proof of concept, two different transcription factors were used, a transcriptional inducer, AlgR, and a repressor, LexA, from Pseudomonas aeruginosa. After the promoters were incubated with the transcription factors, the plasmid was transcribed into RNA and reverse transcribed to cDNA. Gene expression was measured using qRTPCR. Using the ReViTA plasmid, transcription induction of 55% was observed when AlgR protein was added and a 27% transcription reduction with the repressor LexA, compared with the samples without transcription factors. The results demonstrated the correct functioning of ReViTA as a novel method to study transcription factors and gene expression. Thus, ReViTA could be a rapid and accessible in vitro method to evaluate genes and regulators of various species.Crown Copyright © 2023. Published by Elsevier B.V. All rights reserved

Modification of a PE/PPE substrate pair reroutes an Esx substrate pair from the mycobacterial ESX-1 type VII secretion system to the ESX-5 system

Bacterial type VII secretion systems secrete a wide range of extracellular proteins that play important roles in bacterial viability and in interactions of pathogenic mycobacteria with their hosts. Mycobacterial type VII secretion systems consist of five subtypes, ESX-1-5, and have four substrate classes, namely, Esx, PE, PPE, and Esp proteins. At least some of these substrates are secreted as heterodimers. Each ESX system mediates the secretion of a specific set of Esx, PE, and PPE proteins, raising the question of how these substrates are recognized in a system-specific fashion. For the PE/PPE heterodimers, it has been shown that they interact with their cognate EspG chaperone and that this chaperone determines the designated secretion pathway. However, both structural and pulldown analyses have suggested that EspG cannot interact with the Esx proteins. Therefore, the determining factor for system specificity of the Esx proteins remains unknown. Here, we investigated the secretion specificity of the ESX-1 substrate pair EsxB_1/EsxA_1 in Mycobacterium marinum Although this substrate pair was hardly secreted when homologously expressed, it was secreted when co-expressed together with the PE35/PPE68_1 pair, indicating that this pair could stimulate secretion of the EsxB_1/EsxA_1 pair. Surprisingly, co-expression of EsxB_1/EsxA_1 with a modified PE35/PPE68_1 version that carried the EspG5 chaperone-binding domain, previously shown to redirect this substrate pair to the ESX-5 system, also resulted in redirection and co-secretion of the Esx pair via ESX-5. Our results suggest a secretion model in which PE35/PPE68_1 determines the system-specific secretion of EsxB_1/EsxA_1

Entendiendo la síntesis de ADN en patógenos bacterianos: nuevas estrategias para el tratamiento de enfermedades infecciosas

[spa] Las ribonucleótido reductasas (RNR) son enzimas esenciales que convierten los ribonucleótidos en desoxirribonucleótidos, los monómeros utilizados en la síntesis y reparación del ADN. Existen tres clases de RNR (clase I, II y III), que se clasifican en función de su estructura, mecanismo para formar el radical, cofactores que utilizan y requisitos específicos de oxígeno. El genoma de los organismos eucariotas codifica únicamente para la ribonucleótido reductasa de clase Ia, pero hay microorganismos cuyo genoma codifica para más de una RNR. Por ejemplo, el genoma de Pseudomonas aeruginosa contiene información para producir las tres clases de RNR, lo que le aporta una gran ventaja para adaptarse a distintos ambientes. P. aeruginosa es una bacteria Gram-negativa que puede crecer formando una biopelícula (biofilm) donde hay un gradiente de la concentración de oxígeno. P. aeruginosa forma biofilms en tejidos del organismo y dispositivos quirúrgicos, y también se ha encontrado en biofilms polimicrobianos en heridas. Las infecciones por biofilms se consideran infecciones crónicas y son difíciles de tratar, lo que conlleva un mal pronóstico. Esto, sumado a que las terapias actuales son ineficaces, pone de manifiesto la necesidad de encontrar nuevos enfoques terapéuticos para tratar eficazmente las infecciones por biofilms. En nuestro estudio nos centramos en el desarrollo de nuevas terapias para tratar las infecciones por biofilm. En la búsqueda de nuevas dianas moleculares, estudiamos los factores de transcripción AlgR y NrdR. En esta tesis investigamos la relación entre AlgR, clave en la formación del biofilm, y las RNR de P. aeruginosa. Hemos demostrado que AlgR en su estado no fosforilado activa la transcripción de las RNR bajo condiciones de estrés oxidativo cuando P. aeruginosa crece en forma planctónica y formando un biofilm. También determinamos que la RNR de clase II, NrdJ, es esencial durante la infección en el modelo in vivo Galleria mellonella. Asimismo, estudiamos el mecanismo de activación del factor de transcripción NrdR en P. aeruginosa y Escherichia coli. En esta tesis determinamos que NrdR regula los promotores de todas las RNR en P. aeruginosa y E. coli. Según nuestros estudios, NrdR se encontraría formando poblaciones oligoméricas dinámicas en función del cofactor nucleotídico que se le una. NrdR forma grandes estructuras oligoméricas que son inactivas cuando se une ATP y estructuras entre tetrámeros y octámeros que son activas y reprimen la transcripción de las RNR cuando se une dATP. También hemos desarrollado una nueva técnica de biología molecular, llamada ReViTA, que se basa en la transcripción in vitro para medir la expresión de genes cuando están regulados por factores de transcripción, y que permite estudiar la función biológica del factor de transcripción de interés. Finalmente, estudiamos la formación de biofilms polimicrobianos de P. aeruginosa y Staphylococcus aureus en un modelo de herida in vitro para identificar nuevos tratamientos. Analizamos varias terapias que combinan antibióticos y enzimas disgregadoras de la matriz del biofilm solubles o inmovilizadas en nanopartículas. Los resultados apuntan a que, dentro del biofilm, las bacterias de S. aureus están protegidas y que las terapias que combinan enzimas y antibióticos mejoran el tratamiento del biofilm. En conclusión, las RNR y los factores de transcripción que las regulan son posibles dianas moleculares para desarrollar nuevos enfoques terapéuticos contra las infecciones por biofilm. La técnica ReViTA puede ser útil en la identificación de nuevos factores de transcripción y en la evaluación de su papel en la regulación génica. El estudio de biofilms polimicrobianos es fundamental para entender la interacción entre diferentes especies bacterianas y desarrollar tratamientos efectivos contra las infecciones crónicas.[eng] Ribonucleotide reductases (RNR) are enzymes that convert ribonucleotides into deoxyribonucleotides, which are essential for DNA synthesis and repair. RNR are classified in three classes (I, II, and III) based on their structure, mechanism of radical formation, cofactor, and oxygen requirement. Pseudomonas aeruginosa is a Gram-negative bacterium that synthesizes all three classes of RNR, providing a significant adaptive advantage in different environmental conditions. Moreover, P. aeruginosa can form a biofilm. Biofilm infections are chronic and challenging to treat, resulting in severe damage to the host and poor prognosis. Since current therapies are ineffective, new therapeutic approaches are necessary to effectively treat biofilm infections. In this thesis, we focused on the development of new therapies for infectious diseases, particularly biofilm infections. We investigated the relationship between AlgR, a key transcription factor in biofilm formation, and RNR in P. aeruginosa. We demonstrated that AlgR in its non-phosphorylated state activates RNR transcription under oxidative conditions, and that the class II RNR, NrdJ, is essential during Galleria mellonella infection. Additionally, we studied the role of the transcription factor NrdR in P. aeruginosa and Escherichia coli. We have also developed a novel molecular biology technique, called ReViTA, which utilizes in vitro transcription to measure gene expression when the genes are regulated by transcription factors, enabling the study of the biological function of the transcription factor of interest. Finally, we investigated the formation of polymicrobial biofilms by P. aeruginosa and Staphylococcus aureus in an in vitro wound model to identify new treatments. We analyzed several therapies combining antibiotics and biofilm matrix-disintegrating enzymes, either soluble or immobilized in nanoparticles. The results suggest that biofilm disaggregation is best achieved with combination therapies that include enzymes and antibiotics. In conclusion, RNR and the transcription factors that regulate they expression are critical targets for developing new therapeutic approaches against biofilm infections. The ReViTA technique may be useful in identifying novel transcription factors and assessing their role in gene regulation. The study of polymicrobial biofilms is essential for understanding the interaction between different bacterial species and developing effective treatments against chronic infections

3D spatial organization and improved antibiotic treatment of a Pseudomonas aeruginosaStaphylococcus aureus wound biofilm by nanoparticle enzyme delivery

Chronic wounds infected by Pseudomonas aeruginosa and Staphylococcus aureus are a relevant health problem worldwide because these pathogens grow embedded in a network of polysaccharides, proteins, lipids, and extracellular DNA, named biofilm, that hinders the transport of antibiotics and increases their antimicrobial tolerance. It is necessary to investigate therapies that improve the penetrability and efficacy of antibiotics. In this context, our main objectives were to study the relationship between P. aeruginosa and S. aureus and how their relationship can affect the antimicrobial treatment and investigate whether functionalized silver nanoparticles can improve the antibiotic therapy. We used an optimized in vitro wound model that mimics an in vivo wound to co-culture P. aeruginosa and S. aureus biofilm. The in vitro wound biofilm was treated with antimicrobial combinatory therapies composed of antibiotics (gentamycin and ciprofloxacin) and biofilm-dispersing free or silver nanoparticles functionalized with enzymes (α-amylase, cellulase, DNase I, or proteinase K) to study their antibiofilm efficacy. The interaction and colocalization of P. aeruginosa and S. aureus in a wound-like biofilm were examined and detailed characterized by confocal and electronic microscopy. We demonstrated that antibiotic monotherapy is inefficient as it differentially affects the two bacterial species in the mixed biofilm, driving P. aeruginosa to overcome S. aureus when using ciprofloxacin and the contrary when using gentamicin. In contrast, dual-antibiotic therapy efficiently reduces both species while maintaining a balanced population. In addition, DNase I nanoparticle treatment had a potent antibiofilm effect, decreasing P. aeruginosa and S. aureus viability to 0.017 and 7.7%, respectively, in combined antibiotics. The results showed that using nanoparticles functionalized with DNase I enhanced the antimicrobial treatment, decreasing the bacterial viability more than using the antibiotics alone. The enzymes α-amylase and cellulase showed some antibiofilm effect but were less effective compared to the DNase I treatment. Proteinase K showed insignificant antibiofilm effect. Finally, we proposed a three-dimensional colocalization model consisting of S. aureus aggregates within the biofilm structure, which could be associated with the low efficacy of antibiofilm treatments on bacteria. Thus, designing a clinical treatment that combines antibiofilm enzymes and antibiotics may be essential to eliminating chronic wound infections

Data_Sheet_1_3D spatial organization and improved antibiotic treatment of a Pseudomonas aeruginosa–Staphylococcus aureus wound biofilm by nanoparticle enzyme delivery.PDF

Chronic wounds infected by Pseudomonas aeruginosa and Staphylococcus aureus are a relevant health problem worldwide because these pathogens grow embedded in a network of polysaccharides, proteins, lipids, and extracellular DNA, named biofilm, that hinders the transport of antibiotics and increases their antimicrobial tolerance. It is necessary to investigate therapies that improve the penetrability and efficacy of antibiotics. In this context, our main objectives were to study the relationship between P. aeruginosa and S. aureus and how their relationship can affect the antimicrobial treatment and investigate whether functionalized silver nanoparticles can improve the antibiotic therapy. We used an optimized in vitro wound model that mimics an in vivo wound to co-culture P. aeruginosa and S. aureus biofilm. The in vitro wound biofilm was treated with antimicrobial combinatory therapies composed of antibiotics (gentamycin and ciprofloxacin) and biofilm-dispersing free or silver nanoparticles functionalized with enzymes (α-amylase, cellulase, DNase I, or proteinase K) to study their antibiofilm efficacy. The interaction and colocalization of P. aeruginosa and S. aureus in a wound-like biofilm were examined and detailed characterized by confocal and electronic microscopy. We demonstrated that antibiotic monotherapy is inefficient as it differentially affects the two bacterial species in the mixed biofilm, driving P. aeruginosa to overcome S. aureus when using ciprofloxacin and the contrary when using gentamicin. In contrast, dual-antibiotic therapy efficiently reduces both species while maintaining a balanced population. In addition, DNase I nanoparticle treatment had a potent antibiofilm effect, decreasing P. aeruginosa and S. aureus viability to 0.017 and 7.7%, respectively, in combined antibiotics. The results showed that using nanoparticles functionalized with DNase I enhanced the antimicrobial treatment, decreasing the bacterial viability more than using the antibiotics alone. The enzymes α-amylase and cellulase showed some antibiofilm effect but were less effective compared to the DNase I treatment. Proteinase K showed insignificant antibiofilm effect. Finally, we proposed a three-dimensional colocalization model consisting of S. aureus aggregates within the biofilm structure, which could be associated with the low efficacy of antibiofilm treatments on bacteria. Thus, designing a clinical treatment that combines antibiofilm enzymes and antibiotics may be essential to eliminating chronic wound infections.</p

EspH is a hypervirulence factor for Mycobacterium marinum and essential for the secretion of the ESX-1 substrates EspE and EspF

The pathogen Mycobacterium tuberculosis employs a range of ESX-1 substrates to manipulate the host and build a successful infection. Although the importance of ESX-1 secretion in virulence is well established, the characterization of its individual components and the role of individual substrates is far from complete. Here, we describe the functional characterization of the Mycobacterium marinum accessory ESX-1 proteins EccA1, EspG1 and EspH, i.e. proteins that are neither substrates nor structural components. Proteomic analysis revealed that EspG1 is crucial for ESX-1 secretion, since all detectable ESX-1 substrates were absent from the cell surface and culture supernatant in an espG1 mutant. Deletion of eccA1 resulted in minor secretion defects, but interestingly, the severity of these secretion defects was dependent on the culture conditions. Finally, espH deletion showed a partial secretion defect; whereas several ESX-1 substrates were secreted in normal amounts, secretion of EsxA and EsxB was diminished and secretion of EspE and EspF was fully blocked. Interaction studies showed that EspH binds EspE and therefore could function as a specific chaperone for this substrate. Despite the observed differences in secretion, hemolytic activity was lost in all M. marinum mutants, implying that hemolytic activity is not strictly correlated with EsxA secretion. Surprisingly, while EspH is essential for successful infection of phagocytic host cells, deletion of espH resulted in a significantly increased virulence phenotype in zebrafish larvae, linked to poor granuloma formation and extracellular outgrowth. Together, these data show that different sets of ESX-1 substrates play different roles at various steps of the infection cycle of M. marinum